CN105154500B - It is a kind of produce proinsulin human Pichia pastoris fermentation medium and application - Google Patents
It is a kind of produce proinsulin human Pichia pastoris fermentation medium and application Download PDFInfo
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Abstract
The present invention relates to a kind of Pichia pastoris fermentation mediums for producing proinsulin human, the culture medium prescription are as follows: 1~20g/L of peptone, soybean powder 1~10g/L, 85% 3~14ml/L of phosphatase 11,0.3~0.4g/L of calcium carbonate, 7.0~8.0g/L of epsom salt, 6~10g/L of ammonium sulfate, 1.5~2.5g/L of potassium hydroxide, glycerol 40.0g/L, PTM1 solution 4.35ml/L, pH are 5~6；The invention also includes the fermentation process for producing proinsulin human using Pichia pastoris based on above-mentioned culture medium, and using this method, proinsulin human's expression quantity can be improved 43%, and fermentation time be greatly decreased 24~42 hours, obtain yield more higher than conventional method.
Description
Technical field
The invention belongs to technical field of microbial fermentation, specifically, producing finishing for proinsulin human the present invention relates to a kind of
Red yeast fermentation medium and the method for producing proinsulin human using the culture medium.
Background technique
Insulin is maximally efficient Remedies for diabetes, and the method for producing actrapid monotard is divided into chemical synthesis and base
Because of recombination method, method of gene recombination first produces proinsulin human, then carries out subsequent technique and be changed into actrapid monotard or actrapid monotard's class
Like object.Method of gene recombination mainly uses two methods to produce proinsulin human, and a kind of method uses prokaryotes Escherichia coli, separately
A kind of method uses eucaryote saccharomyces cerevisiae.
Pichia pastoris both had the breeding of similar prokaryotes Escherichia coli it is fast, easily culture, convenient for the characteristic of genetic manipulation,
Again the albumen with eucaryote saccharomyces cerevisiae can the characteristic that correctly folds of posttranslational modification, albumen, application is very extensive, Bi Chi
Yeast have become multiple protein ideal expression system (Lin Cereghino GP, Lin Cereghino J, Ilgen C,
Cregg J M(2002)Production of recombinant proteins in fermenter cultures ofthe
yeast Pichia pastoris.Curr Opin Biotechnol 13:329–332)。
It needs in Pichia anomala expression foreign protein using the nutritional ingredient in culture medium, culture medium is in addition to that must contain bacterium
Outside substance necessary to body growth metabolism and element, it is necessary to contain substance and element required for composition foreign protein, e.g., carbon
Source, nitrogen source, inorganic salts etc.；Carbon source is mainly to provide the carbon skeleton of cell and product to the effect that yeast growth is metabolized, and provides thin
Energy needed for born of the same parents' vital movement, carbon source can be divided into inorganic carbon source material and organic carbon source substance, including glucose, fructose,
The carbohydrates such as sucrose, starch, cellulose further include organic acid, amino acid, alcohols, aldehyde, the carbon compounds such as phenol；Pichia pastoris is normal
The carbon source utilized is generally glycerol, glucose, methanol etc..Nitrogen source provides for yeast cells and constitutes protein, nucleic acid and other nitrogen
The material of element compound, nitrogen source can be divided into inorganic nitrogen-sourced and organic nitrogen source, and inorganic nitrogen-sourced includes ammonium salt, nitrate etc., is had
Machine nitrogen source includes beef extract, peptone, yeast extract, fish meal, blood meal, cicada pupa powder, beans (cake) powder, peanut (cake) powder etc..
Pichia pastoris fermentation, which produces proinsulin human, can use a variety of culture mediums, generally on the basis of BSM culture medium and BSM
Improved culture medium.BSM (basic salt) culture medium is the conventional use of training of Pichia pastoris fermentation that Invitrogen company provides
Support base, formula are as follows: 85% phosphoric acid 26.7ml/L, calcium sulphate dihydrate 0.93g/L, potassium sulfate 18.2g/L, epsom salt
14.9g/L, potassium hydroxide 4.13g/L, glycerol 40.0g/L；Dong Peng etc. carries out fermentation tank culture using BSM culture medium and produces people's pancreas islet
Plain former, 30 DEG C of inducing temperature, induction pH is 5.5~6.0, induction 96h, expression quantity 256mg/L (Dong Peng etc., expression of insulin
Pichia pastoris growth kinetics and induction strategies.Beijing University of Chemical Technology's journal, 2006,33 (3): 37~41)；País-
Chanfrau etc. adds casein hydrolysate, yeast extract, peptone and ammonium sulfate for expressing respectively in BSM culture medium
Proinsulin 22 DEG C of inducing temperature, induces pH 6.3, expression quantity after 120h is induced to reach 0.3g/L (Pa í s-Chanfrau J
M,et al.Improving the expression ofmini-proinsulin inPichia
pastoris.BiotechnolLett.,2004,26(16):1269-1272)；Gurramkonda etc. will be in BSM culture medium
Calcium sulphate dihydrate 0.93g/L is changed to CALCIUM CHLORIDE DIHYDRATE 0.28g/L, and phosphoric acid and potassium hydroxide are changed to potassium dihydrogen phosphate 9.4g/
L, glycerol content increase to 95.2g/L, and are added to the ammonium sulfate of 15.7g/L, carry out human insulin precursor using this culture medium
Fermentation, 30 DEG C of inducing temperature, induction pH is 5.5, and inducing expression 120h, expression quantity reaches 3-4g/L (Gurramkonda et
al.Application of simple fed-batch technique to high-level secretory
production ofinsulin precursor using Pichia pastoris with subsequent
purification and conversion to human insulin.Microbial Cell Factories 2010,9:
31~41).
Perhaps expression quantity is not high or Fiber differentiation overlong time for above-mentioned culture medium, causes production efficiency not high, also,
It is high that some researches show that BSM medium salts concentration, so that culture medium osmotic pressure is excessively high, causes the thallus death rate to increase, not only influences
The raising of expression quantity, release lipid material also add downstream purification technique difficulty (Brady C P, Shimp RL,
MilesAR,et al.High-level production and purification of P30P2MSP1(19),an
important vaccine antigen for malaria,expressed in the methylotropic yeast
Pichia pastoris.Prot Expres Purif.2001,23,468-475), therefore, it is necessary to change to culture medium
Into providing one kind not only can be shortened incubation time, but also can obtain the culture medium of the proinsulin human of high expression quantity, to improve industrialization
The efficiency of production.
Summary of the invention
The present invention is to solve to produce proinsulin human's expression quantity using the conventional use of BSM culture medium fermentation of Pichia pastoris fermentation
The problem that not high, Fiber differentiation overlong time etc. causes production efficiency not high provides a kind of suitable for Pichia pastoris fermentation production people
The culture medium of proinsulin, and on this basis, it ferments the present invention also provides a kind of progress Pichia pastoris and produces proinsulin human
Method.
The purpose of the present invention is realized by following scheme:
A kind of Pichia pastoris fermentation medium producing proinsulin human, the culture medium prescription are as follows: 1~20g/L of peptone, it is yellow
Bean powder 1~10g/L, 85% 3~14ml/L of phosphatase 11,0.3~0.4g/L of calcium carbonate, 7.0~8.0g/L of epsom salt, sulfuric acid
6~10g/L of ammonium, 1.5~2.5g/L of potassium hydroxide, glycerol 40.0g/L, PTM1 solution 4.35ml/L, pH are 5~6.
Preferably, culture medium prescription are as follows: peptone 10g/L, soybean powder 5g/L, 85% phosphatase 11 3ml/L, calcium carbonate
0.33g/L, epsom salt 7.4g/L, ammonium sulfate 8g/L, potassium hydroxide 2g/L, glycerol 40.0g/L, PTM1 solution 4.35ml/
L, pH 5.5.
Preferably, culture medium prescription are as follows: peptone 20g/L, soybean powder 10g/L, 85% phosphatase 11 4ml/L, calcium carbonate
0.4g/L, epsom salt 7g/L, ammonium sulfate 6g/L, potassium hydroxide 2.5g/L, glycerol 40.0g/L, PTM1 solution 4.35ml/L,
PH is 5.5.
The peptone is microbe-derived or Yeast protein peptone.The present invention is according to Invitrogen life
Regulation method in technologies company " Pichia fermentation process guidelines " finish red
The fermented and cultured of yeast.The Pichia pastoris refers to using pichia pastoris yeast (Pichiapastoris) GS115 as expression
The gene recombination bacterium of host, the fermentation are to carry out methanol induction using Pichia pastoris in the fermenter to express foreign protein people
Proinsulin.
A method of Pichia pastoris fermentation is carried out based on culture medium of the present invention and produces proinsulin human, it is specific comprising with
Lower step:
(1) the Pichia pastoris glycerol strain of preservation the preparation of seed liquor: is connected to the 500ml of the culture medium of YPD containing 100ml
Shaking flask, 30 DEG C, 250rpm 19~20h of shaking table culture, as seed liquor；
(2) batch culture fermentations: 1L seed liquor accesses in the 50L fermentor of the fermentation medium containing 19L, originates condition of culture
It is 30 DEG C, pH 5.0, revolving speed 300rpm, ventilatory capacity 0.5m3/h.With the progress of culture, revolving speed and ventilatory capacity are increased accordingly, is controlled
Dissolved oxygen 20~30% processed cultivates 19~21h；
(3) after batch culture, PTM1 containing 12ml/L glycerol feed-batch culture in batches: is added with the rate stream of 10ml/h/L
50% 5~6h of glycerol of solution, revolving speed is adjusted in incubation and ventilatory capacity controls dissolved oxygen 20~30%；
(4) methanol induction culture: in batches after glycerol feed supplement, controlled at 25~28 DEG C, pH is 3.0~5.0, with
After the rate stream of 3.6ml/h/L adds 5~6h of methanol of the solution of PTM1 containing 12ml/L, increase methanol feeding rate is 10ml/h/L,
Until fermentation ends.Revolving speed is adjusted in Induction Process and ventilatory capacity controls dissolved oxygen 20~30%；
(5) sampling and fermentation ends: after methanol induction culture starts, taking fermentation broth sample primary in every 3~12 hours, and 3000
~12000rpm is centrifuged 5~10min, measures proinsulin human's expression quantity in supernatant, when amount to be expressed increases slowly or reduces,
Terminate fermentation.
The YPD culture medium of step (1) is prepared by following methods: yeast powder 10g/L, tryptone 20g/L, glucose 20g/
L, 121 DEG C of sterilizing 20min.
The temperature of step (4) is preferably 25 DEG C.
The pH of step (4) is preferably 3.25.
The present invention has the following advantages and beneficial effects: compared with the existing technology
Culture medium provided by the invention produces proinsulin human for Pichia pastoris fermentation, normal relative to Pichia pastoris fermentation
Other culture mediums for advising the BSM culture medium used and prior art offer, can be more effectively carried out Pichia pastoris fermenting and producing
Proinsulin human.Based on culture medium provided by the invention, is fermented using Pichia pastoris provided by the invention and produce proinsulin human's
Method, proinsulin human's expression quantity greatly improve, and up to 43%, and 24~42h of incubation time are greatly decreased, to improve life
Produce efficiency.
Detailed description of the invention
Fig. 1 is the curve graph that proinsulin human's expression quantity changes with induction time in 1 control experiment of embodiment.
Fig. 2 is the curve graph that proinsulin human's expression quantity changes with induction time in embodiment 2.
Fig. 3 is the curve graph that proinsulin human's expression quantity changes with induction time in embodiment 8.
Specific embodiment
The present invention is further illustrated below by way of specific embodiment, embodiment is merely to illustrate the present invention without limiting this hair
Bright range claimed.
Embodiment 1: control experiment
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment is BSM culture medium, formula are as follows: 85% phosphoric acid
26.7ml/L, calcium sulphate dihydrate 0.93g/L, potassium sulfate 18.2g/L, epsom salt 14.9g/L, potassium hydroxide 4.13g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L are 5.5 with potassium hydroxide and phosphorus acid for adjusting pH.
Use Invitrogen life technologies company " Pichia fermentation process
Guidelines " in provide Pichia pastoris fermentation routine techniques, in accordance with the following steps carry out Pichia pastoris fermenting and producing people
Proinsulin:
(1) the Pichia pastoris glycerol strain of preservation the preparation of seed liquor: is connected to the 500ml of the culture medium of YPD containing 100ml
Shaking flask, 30 DEG C, 250rpm shaking flask culture 20h, as seed liquor.
(2) batch culture fermentations: 1L seed liquor accesses in the 50L fermentor of the fermentation medium containing 19L, originates condition of culture
It is 30 DEG C, pH 5.0, revolving speed 300rpm, ventilatory capacity 0.5m3/h.With the progress of culture, revolving speed and ventilatory capacity are increased accordingly, is controlled
Dissolved oxygen 20~30% processed cultivates 20h.
(3) after batch culture, PTM1 containing 12ml/L glycerol feed-batch culture in batches: is added with the rate stream of 10ml/h/L
50% 5~6h of glycerol of solution, revolving speed is adjusted in incubation and ventilatory capacity controls dissolved oxygen 20~30%.
(4) methanol induction culture: in batches after glycerol feed supplement, inducing temperature is reduced to 25 DEG C, and induction pH is 3.25, with
After the rate stream of 3.6ml/h/L adds the methanol 6h of the solution of PTM1 containing 12ml/L, increase methanol feeding rate is 10ml/h/L, directly
To fermentation ends.Revolving speed is adjusted in Induction Process and ventilatory capacity controls dissolved oxygen 20~30%.
(5) sampling and fermentation ends: after methanol induction culture starts, take fermentation broth sample primary within every 12 hours, wait induce
Every 3~6h sampling is primary after expressing 84h, and 12000rpm is centrifuged 5min, measures proinsulin human's expression quantity in supernatant, to be expressed
When amount increases slowly or reduces, terminate fermentation.
When testing result shows to induce 96h, the expression quantity of proinsulin human reaches maximum value in fermented supernatant fluid
2571mg/L (Fig. 1).
Embodiment 2
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 1g/L, soybean powder 1g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.3g/L, epsom salt 7.0g/L, ammonium sulfate 6g/L, potassium hydroxide 1.5g/L, glycerol
40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.0, and sample time is primary for 3~6h sampling every after inducing expression 48h in step (5), other are the same as implementation
Example 1.When testing result shows to induce 57h, the expression quantity of proinsulin human reaches maximum value 2682mg/L in fermented supernatant fluid
(Fig. 2) is compared with control experiment, and incubation time reduces 39h, and expression quantity improves 4.3%.
Embodiment 3
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 10g/L, soybean powder 1g/L,
85% phosphatase 11 3.3ml/L, calcium carbonate 0.4g/L, epsom salt 8.0g/L, ammonium sulfate 6.0g/L, potassium hydroxide 2.5g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 28 DEG C, induction pH is 5.0, and step (5) is the same as embodiment 2.When testing result shows to induce 54h, people's pancreas islet in fermented supernatant fluid
Plain former expression quantity reaches maximum value 2580mg/L and compares with control experiment, and incubation time reduces 42h, and expression quantity improves
0.3%.
Embodiment 4
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 10g/L, soybean powder 1g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.3g/L, epsom salt 7.0g/L, ammonium sulfate 6.0g/L, potassium hydroxide 1.5g/L, glycerol
40.0g/L, PTM1 solution 4.35ml/L, pH are 5~6.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 26 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 1.When testing result shows to induce 60h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3278mg/L and compares with control experiment, and incubation time reduces 36h, and expression quantity improves
27.4%.
Embodiment 5
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 1g/L, soybean powder 10g/L,
85% phosphatase 11 4ml/L, calcium carbonate 0.4g/L, epsom salt 7.0g/L, ammonium sulfate 10.0g/L, potassium hydroxide 1.5g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 27 DEG C, induction pH is 5.0, and step (5) is the same as embodiment 2.When testing result shows to induce 55h, people's pancreas islet in fermented supernatant fluid
Plain former expression quantity reaches maximum value 2808mg/L and compares with control experiment, and incubation time reduces 41h, and expression quantity improves
9.2%.
Embodiment 6
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 1g/L, soybean powder 10g/L,
85% phosphatase 11 4ml/L, calcium carbonate 0.3g/L, epsom salt 8.0g/L, ammonium sulfate 10.0g/L, potassium hydroxide 2.5g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 2.When testing result shows to induce 57h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3137mg/L and compares with control experiment, and incubation time reduces 39h, and expression quantity improves
22%.
Embodiment 7
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 10g/L, soybean powder 5g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.33g/L, epsom salt 7.4g/L, ammonium sulfate 10.0g/L, potassium hydroxide 2.0g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 26 DEG C, induction pH is 4, and step (5) is the same as embodiment 2.When testing result shows to induce 60h, actrapid monotard in fermented supernatant fluid
Former expression quantity reaches maximum value 3390mg/L and compares with control experiment, and incubation time reduces 36h, and expression quantity improves 31.8%.
Embodiment 8
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 10g/L, soybean powder 5g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.33g/L, epsom salt 7.4g/L, ammonium sulfate 8.0g/L, potassium hydroxide 2.0g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 2.When testing result shows to induce 63h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3678mg/L (Fig. 3) and compares with control experiment, and incubation time reduces 33h, and expression quantity mentions
It is high by 43%.
Embodiment 9
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 10g/L, soybean powder 10g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.33g/L, epsom salt 7.4g/L, ammonium sulfate 6.0g/L, potassium hydroxide 2.0g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.5, and step (5) is the same as embodiment 2.When testing result shows to induce 66h, people's pancreas islet in fermented supernatant fluid
Plain former expression quantity reaches maximum value 3435mg/L and compares with control experiment, and incubation time reduces 30h, and expression quantity improves 33%.
Embodiment 10
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 20g/L, soybean powder 10g/L,
85% phosphatase 11 4ml/L, calcium carbonate 0.4g/L, epsom salt 7.0g/L, ammonium sulfate 6.0g/L, potassium hydroxide 2.5g/L, glycerol
40.0g/L, PTM1 solution 4.35ml/L, pH .5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 2.When testing result shows to induce 72h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3544mg/L and compares with control experiment, and incubation time is reduced for 24 hours, and expression quantity improves
37%.
Embodiment 11
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 20g/L, soybean powder 1g/L,
85% phosphatase 11 3ml/L, calcium carbonate 0.4g/L, epsom salt 7.0g/L, ammonium sulfate 6.0g/L, potassium hydroxide 2.5g/L, glycerol
40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 2.When testing result shows to induce 69h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3253mg/L and compares with control experiment, and incubation time reduces 27h, and expression quantity improves
26.5%.
Embodiment 12
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 20g/L, soybean powder 1g/L,
85% phosphatase 11 3.3ml/L, calcium carbonate 0.3g/L, epsom salt 8.0g/L, ammonium sulfate 10.0g/L, potassium hydroxide 1.5g/L,
Glycerol 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 25 DEG C, induction pH is 3.25, and step (5) is the same as embodiment 2.When testing result shows to induce 69h, people's pancreas in fermented supernatant fluid
Element former expression quantity in island reaches maximum value 3499mg/L and compares with control experiment, and incubation time reduces 27h, and expression quantity improves
36%.
Embodiment 13
The Pichia pastoris fermentation medium of the production proinsulin human of the present embodiment are as follows: peptone 20g/L, soybean powder 10g/L,
85% phosphatase 11 4ml/L, calcium carbonate 0.4g/L, epsom salt 8.0g/L, ammonium sulfate 10.0g/L, potassium hydroxide 2.5g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH 5.5.
The step of Pichia pastoris fermenting and producing proinsulin human (1)~(3) are with embodiment 1, inducing temperature drop in step (4)
It is 28 DEG C, induction pH is 5.0, and step (5) is the same as embodiment 2.When testing result shows to induce 69h, people's pancreas islet in fermented supernatant fluid
Plain former expression quantity reaches maximum value 3054mg/L and compares with control experiment, and incubation time reduces 27h, and expression quantity improves
18.7%.
Table 1 is each embodiment and culture medium prescription and expression quantity result in control experiment.It is in table the result shows that: this hair
The culture medium of bright middle offer reduces culture by changing component of inorganic salts or concentration relative to conventional use of BSM culture medium
Total salt content in base, and the required nitrogen source of Pichia pastoris fermentation is added, incubation time is not only shortened, but also high expression quantity can be obtained
Proinsulin human, improve the efficiency of production.
Each embodiment of table one and culture medium prescription and expression quantity Comparative result in control experiment
Claims (6)
1. it is a kind of using fermentation medium carry out Pichia pastoris fermentation produce proinsulin human method, which is characterized in that including with
Lower step:
(1) the Pichia pastoris glycerol strain of preservation: being connected to the 500ml shaking flask of the culture medium of YPD containing 100ml by the preparation of seed liquor,
30 DEG C, 250rpm 19~20h of shaking table culture, as seed liquor；
(2) batch culture fermentations: 1L seed liquor accesses in the 50L fermentor of the fermentation medium containing 19L, and starting condition of culture is 30
DEG C, pH 5.0, revolving speed 300rpm, ventilatory capacity 0.5m3/ h increases accordingly revolving speed and ventilatory capacity with the progress of culture, controls molten
Oxygen 20~30% cultivates 19~21h；
(3) after batch culture, the solution of PTM1 containing 12ml/L glycerol feed-batch culture in batches: is added with the rate stream of 10ml/h/L
50% 5~6h of glycerol, revolving speed is adjusted in incubation and ventilatory capacity controls dissolved oxygen 20~30%；
(4) methanol induction culture: in batches after glycerol feed supplement, inducing temperature is reduced to 25~28 DEG C, and induction pH is adjusted to 3.25, with
After the rate stream of 3.6ml/h/L adds 5~6h of methanol of the solution of PTM1 containing 12ml/L, increase methanol feeding rate is 10ml/h/L,
Dissolved oxygen 20~30% is controlled up to adjusting revolving speed and ventilatory capacity in fermentation ends, Induction Process；
(5) sampling and fermentation ends: after methanol induction culture starts, taking fermentation broth sample primary in every 3~12 hours, 3000~
12000rpm is centrifuged 5~10min, measures proinsulin human's expression quantity in supernatant, when amount to be expressed increases slowly or reduces, knot
Beam fermentation；
The formula of the fermentation medium are as follows: 1~20g/L of peptone, soybean powder 1~10g/L, 85% 3~14ml/L of phosphatase 11,
0.3~0.4g/L of calcium carbonate, 7.0~8.0g/L of epsom salt, 6~10g/L of ammonium sulfate, potassium hydroxide 1.5~2.5g/L are sweet
Oily 40.0g/L, PTM1 solution 4.35ml/L, pH are 5~6.
2. according to claim 1 carry out the method that Pichia pastoris fermentation produces proinsulin human using fermentation medium,
It is characterized in that, the formula of the fermentation medium are as follows: peptone 10g/L, soybean powder 5g/L, 85% phosphatase 11 3ml/L, calcium carbonate
0.33g/L, epsom salt 7.4g/L, ammonium sulfate 8g/L, potassium hydroxide 2g/L, glycerol 40.0g/L, PTM1 solution 4.35ml/
L, pH 5.5.
3. according to claim 1 carry out the method that Pichia pastoris fermentation produces proinsulin human using fermentation medium,
It is characterized in that, the formula of the fermentation medium are as follows: peptone 20g/L, soybean powder 10g/L, 85% phosphatase 11 4ml/L, calcium carbonate
0.4g/L, epsom salt 7g/L, ammonium sulfate 6g/L, potassium hydroxide 2.5g/L, glycerol 40.0g/L, PTM1 solution 4.35ml/L,
PH is 5.5.
4. according to claim 1-3 carry out Pichia pastoris fermentation production proinsulin human's using fermentation medium
Method, which is characterized in that the peptone is microprotein peptone or Yeast protein peptone.
5. according to claim 1 carry out the method that Pichia pastoris fermentation produces proinsulin human using fermentation medium,
It is characterized in that, inducing temperature is 25 DEG C in step (4).
6. according to claim 1 carry out the method that Pichia pastoris fermentation produces proinsulin human using fermentation medium,
It is characterized in that, genetic recombination of the Pichia pastoris using pichia pastoris yeast (Pichia pastoris) GS115 as expressive host
Bacterium, the fermentation are to carry out methanol induction using Pichia pastoris in the fermenter to express foreign protein proinsulin human.
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| CN111704999A (en) * | 2020-07-01 | 2020-09-25 | 宁夏西麒麟生物科技有限公司 | Method for preparing culture medium by enzymolysis of organic raw materials by using enzymatic degradation technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139626A (en) * | 2006-09-06 | 2008-03-12 | 上海新生源生物医药有限公司 | Method for producing human insulinogen C peptide |
| CN101372705A (en) * | 2008-07-04 | 2009-02-25 | 上海欣百诺生物科技有限公司 | Preparation of recombinant long-acting glucagon peptide analogue |
| CN103388015A (en) * | 2013-07-24 | 2013-11-13 | 甘肃华羚生物技术研究中心 | Preparation method of yak milk casein anticoagulant peptide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139626A (en) * | 2006-09-06 | 2008-03-12 | 上海新生源生物医药有限公司 | Method for producing human insulinogen C peptide |
| CN101372705A (en) * | 2008-07-04 | 2009-02-25 | 上海欣百诺生物科技有限公司 | Preparation of recombinant long-acting glucagon peptide analogue |
| CN103388015A (en) * | 2013-07-24 | 2013-11-13 | 甘肃华羚生物技术研究中心 | Preparation method of yak milk casein anticoagulant peptide |
Non-Patent Citations (3)
| Title |
|---|
| 《RAespeaprchlication of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin》;Gurramkonda C. 等;《Microbial Cell Factories》;20100512;第9卷(第1期);参见第8页左栏第二段 |
| 《Secretory Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin》;T. Xie 等;《Preparative Biochemistry & Biotechnology》;20080617;第38卷(第31期);第310页第3段 |
| 《表达胰岛素的毕赤酵母生长动力学及诱导策略》;董鹏等;《北京化工大学学报》;20060331;第33卷(第3期);全文 |
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